Imaging system

ABSTRACT

A method of investigating the location and size of a light-emitting source in a subject is disclosed. In practicing the method, one first obtains a light intensity profile by measuring, from a first perspective with a photodetector device, photons which (i) originate from the light-emitting source, (ii) travel through turbid biological tissue of the subject, and (iii) are emitted from a first surface region of interest of the subject. The light-intensity profile is matched against with a parameter-based biophotonic function, to estimate function parameters such as depth and size. The parameters so determined are refined using data other than the first measured light intensity profile, to obtain an approximate depth and size of the source in the subject. Also disclosed is an apparatus for carrying out the method.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to commonlyowned and co-pending U.S. patent application Ser. No. 12/823,019, filedon Jun. 24, 2010, which claims priority to U.S. Pat. No. 7,764,986,which in turn claims priority to U.S. Pat. No. 7,403,812, all of whichare entitled, “METHOD AND APPARATUS FOR DETERMINING TARGET DEPTH,BRIGHTNESS AND SIZE WITHIN A BODY REGION,” and the latter of whichclaims priority to Provisional Application Ser. No. 60/291,794, filed onMay 17, 2001; each of these patent applications is incorporated byreference herein in its entirety for all purposes.

FIELD

This invention relates to an apparatus and method for fields ofnon-invasive medical imaging, medical research, pathology, and drugdiscovery and development.

BACKGROUND

In a variety of medical diagnostic and therapeutic settings, as well asin biomedical research, it is desirable to image a subsurface target orarea within a body region of a subject. For example, non-invasivelocating and imaging of part or all of a solid tumor, an area ofmyocardial ischemia, the distribution of a therapeutic compoundadministered to the subject, or the progression of a disease may provideuseful research or diagnostic information. Ideally, an imaging method isable to locate within a body region a target of interest, and provideinformation about the target's shape, size, number of cells, and depthbelow the surface of the body region. However, until now, methods thathave been used and/or proposed for subsurface body target imaging havegenerally been limited to those using ionizing radiation such as X-rays,expensive and bulky equipment such as Magnetic Resonance imaging (MRI),or ultra-sound.

X-rays have excellent tissue penetration, and when used in conjunctionwith computed tomography (CT) or computed axial tomography (CAT), canproduce superior image quality. However, X-rays have limited use inmonitoring disease progress because exposure to X-rays is potentiallyharmful if such exposure is prolonged. X-rays can be used to locate andimage compositions that have localized at a target within a body region,but always with exposure to the potential harm associated with X-rayradiation. X-rays, however, cannot readily be used to image theexpression of gene products in vivo and determine the depth and/or shapeof a target expressing such gene products.

MRI is also an excellent method for imaging targets, areas, andstructures in a body region of a subject. Although MRI is not thought topossess harmful attributes like those associated with ionizingradiation, the expense and bulky equipment size needed to use MRI makeit impractical for many applications or situations. MRI can provide twoand three-dimensional information about targets within a body region ofa subject, but is less effective at imaging physiological activityassociated with a target.

Ultrasound or ultrasonography is the use of high-frequency sound(ultrasonic) waves to produce images of structures within the humanbody. Ultrasonic waves are sound waves that are above the range of soundaudible to humans. Ultrasonic waves are produced by the electricalstimulation of a piezoelectric crystal and such waves can be aimed atspecific body regions. As the waves travel through body tissues within abody region, they are reflected back at any point where there is achange in tissue density, as, for instance, in the border between twodifferent organs of the body. Ultrasound offers the advantages of notusing radiation or radioactive material, and employs lesser expensiveand less bulky equipment than MRI, but is limited to only discerningdifferences in density of underlying tissue and structures. Accordingly,ultrasound cannot effectively track and monitor the progress of aninfection unless such infection results in a discernable shift indensity of the target tissue. Ultrasound cannot image or detect thephysiological functions of tissues or organs.

Until now, Positron Emission Tomography or P.E.T. was unique amongimaging techniques because it produces an image of organ or tissuefunction. Other imaging techniques such as X-ray, CT, MRI, andsonography depict organ or tissue anatomy but cannot discernphysiological activity within them. To image a specific biochemicalactivity of an organ, a radioactive substance, called a radiotracer orradiopharmaceutical, is injected into the body or inhaled. The tracer isusually a radioactive equivalent of a substance that occurs naturallywithin the body such as water or sugar. The radioactive isotope isidentical to the body's own nonradioactive isotope except that its atomshave a different number of neutrons. Consequently, a subject's body isburdened with radioactive material, and the potential harm associatedwith such material. P.E.T cannot detect non-isotopic expression productsfrom transgenic tissues, organs, or transgenic organisms. Scintigraphy,a diagnostic technique in which a two-dimensional picture of a bodilyradiation source is obtained by the use of radioisotopes, may also beused for imaging structures and their functions. Scintigraphy, however,is not suitable for determining the depth of a target in a body regionof a subject.

In view of the above-mentioned technologies for locating and imaging atarget in a body region of a subject, there is a need for methods anddevices to determine the depth and/or the shape and/or number of cellsof such target without having to use radioactivity, radiation, orexpensive and bulky equipment. The invention disclosed herein meetsthese needs.

SUMMARY

In one aspect, the invention includes a method of investigating thelocation, size and number of cells, of a light-emitting source in asubject. In practicing the method, one initially obtains a firstmeasured light intensity profile constructed by measuring, from a firstperspective with a photodetector device, photons which (i) originatefrom the light-emitting source, (ii) travel through turbid biologicaltissue of the subject, and (iii) are emitted from a first surface regionof interest of the subject. The light-intensity profile is matchedagainst a parameter-based biophotonic function to estimate functionparameters such as depth and size. The parameters so determined arerefined using data other than the first measured light intensityprofile, to obtain an approximate depth and size of the source in thesubject. The additional data may be data measured from the subject, datafrom modeling analysis, or data relating to the wavelength of photonsemitted from the surface of the subject. As examples:

The method typically includes generating a 2-D or 3-D visualrepresentation of the light-emitting source, using the approximate depthand shape of the source in the subject, and superimposing the visualrepresentation onto a 2-D or 3-D image of the subject.

The additional data may be obtained from computer simulation of thediffusion of light from a light-emitting source in a turbid medium. Onepreferred simulation approach involves (i) generating a plurality oftheoretical light-intensity profiles, based on a model of photondiffusion from a glowing source located at one of a plurality of depths,and having one of a plurality of sizes and shapes, through a turbidmedium having absorption and scattering properties similar to those ofbiological tissue, (ii) comparing the quality of fit between each of theplurality of theoretical light-intensity profiles and the first measuredlight intensity profile, (iii) selecting the theoretical light intensityprofile which provides to the first measured light intensity profile,and (iv) obtaining an approximate depth, shape and brightness of thesource in the subject using parameters from the theoretical lightintensity profile selected in (iii). The method may include employing ina photon-scattering model, one or more predetermined tissue-specificlight-scattering coefficients corresponding to tissue through which thephotons travel.

In another general embodiment, the additional data are obtained bymeasuring light emission from the subject at two or more differentwavelengths between about 400 nm and about 1000 nm, determining therelative light intensities measured at the different wavelengths, andcomparing the determined relative light intensities with known relativesignal intensities at the different wavelengths, as a function of tissuedepth. Alternatively, the spectrum of light intensities is measured overa selected wavelength range between about 400-1000 nm, and the measuredspectrum is compared with a plurality of spectra measured fromlight-emitting sources placed at various depths within tissue, todetermine the depth of the light-emitting source from matching themeasured spectrum with the known spectra.

In various embodiments for a light-emission source, the source is aluminescent moiety or a fluorescent moiety; the light-emitting source isadministered to the subject and binds to a selected target or isotherwise localized in the subject prior to the measuring; and thelight-emitting source is a light-generating protein (e.g., a luciferase,a green fluorescent protein, etc.), such as a light-generating proteinexpressed by biological cells of the subject or biological cellsadministered to the subject.

In another aspect, the invention includes apparatus for use ininvestigating the location and size of a light-emitting source in asubject. The apparatus includes a light-tight enclosure within whichlight-emission events from the subject can be detected and an opticalsystem associated with the enclosure for use in generating a first lightintensity profile constructed by measuring, from a first perspectivewithin the enclosure, photons which (i) originate from thelight-emitting source, (ii) travel through turbid biological tissue ofthe subject, and (iii) are emitted from a first surface region ofinterest of the subject. A computational unit operatively connected tothe photo-detector functions to (i) fit the first measured lightintensity profile with a parameter-based biophotonic function; and (ii)refine the parameters of the biophotonic function, using data other thanthe first measured light intensity profile, to generate an approximatedepth and shape of the source in the subject.

The optical system preferably includes an intensified or cooledcharge-coupled-device (CCD), and a lens for focusing light onto the CCD.The optical system may configured in such a way as to detect photonsemitted from a plurality of different selected surface regions ofinterest of the subject. The system may include one or more filters fortransmitting photons within different selected wavelength ranges, e.g.,above and below 600 nm.

The computational unit may include a data file of model biophotonicfunctions representing light-emitting sources of various sizes atvarious depths, for curve matching with the first spatial profile.

Where the optical system includes filters for wavelength discrimination,the computation unit is operable to carry out at least one of theparameter-refinement operations:

(i) determining the relative light intensities measured at the differentwavelengths, and comparing the determined relative light intensitieswith known or calculated relative signal intensities at the differentwavelengths, as a function of tissue depth; and

(ii) comparing the measured spectrum with a plurality of spectrameasured from light-emitting sources placed at various depths withintissue, and determining the depth of the light-emitting source frommatching the measured spectrum with the known spectra.

In another embodiment, the computational unit is operable to average theintensity pattern image into a single intensity value, and further todetermine source size by integrated light intensity values generated asa function of a light-emitting source of a particular size and shape.

The computational unit may have a database containing a plurality oftheoretical light-intensity profiles, based on a model of photondiffusion from a light-emitting source located at one of a plurality ofdepths, and having one of a plurality of sizes and shapes, through aturbid medium having absorption and scattering properties similar tothose of biological tissue. Here the computational unit is operable to(i) compare the quality of fit between each of the plurality oftheoretical light-intensity profiles and first measured light intensityprofile, (ii) select the theoretical light intensity profile whichprovides the best fit to the first measured light intensity profile, and(iii) obtain an approximate depth and shape of the source in the subjectusing parameters from the theoretical light intensity profile selectedin (ii).

In addition, the computational unit is operable to generate a visual 2-,or 3-dimensional representation of the light-emitting source, using theapproximate depth and shape of the source in the subject, andsuperimposing the visual representation onto a 2- or 3-dimensional imageof the subject.

In another aspect, the invention provides a method of determining thedepth of a light-emitting source in a subject. In practicing the method,the light emission intensity from the subject at two or more differentwavelengths between about 400 and about 1000 nm is measured. The depthof the light-emitting source is determined using the measured lightintensities and information related to the optical properties of thesubject's tissue, e.g., coefficients of attenuation and diffusion in thesubject.

Information relating to optical properties, e.g., the coefficients ofattenuation and diffusion in the subject, may be obtained by directmeasurement of the coefficients in material that is the same or similarto that of the subject.

Alternatively, optical-properties information may be obtainedindirectly, by determining, at two or more different wavelengths, lightintensities from a light-emitting source located at each of a pluralityof depths in tissue or material corresponding to that of the subject.The desired depth determination is then carried out by matching themeasured light intensities with the light intensities determined at eachof the plurality of depths.

In the latter approach, the spectral profile of light intensities fromthe light-emitting source may be compared (matched) with each of aplurality of spectral profiles of light intensities from alight-emitting source at each of a plurality of depths.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cross-sectional view of an apparatus constructed inaccordance with the invention, for use in investigating the location andsize of a light-emitting source in a subject.

FIG. 1B illustrates features of the apparatus in FIG. 1A in schematicform.

FIG. 2 is a flowchart of the general steps that can be carried out bythe apparatus, in practicing the method of the invention.

FIG. 3A is a surface light-intensity image from a light-emitting sourcein an animal subject.

FIG. 3B is a light emission profile from the animal subject in FIG. 3A,showing the curve fit between the measured emission profile (solidlines) and a light-emission profile calculated from a diffusion model.

FIGS. 4A and 4B are plots of the emission spectra of bacterialluciferase in vitro (solid line), and in vivo (dashed lines) at variousdepths in a mouse thigh, as indicated, where the plot in FIG. 4B hasbeen scaled to show the peak intensity in vivo in the region between600-700 nm.

FIG. 5 shows various locations in an animal at which transmissionspectra were obtained.

FIGS. 6A and 6B show transmission spectra through a live experimentalanimal at the various animal locations shown in FIG. 5.

FIG. 6C illustrates a double integrating sphere apparatus useful formeasuring optical properties of a sample, e.g., tissue.

FIG. 6D illustrates the experimental set up of a radial fiber probe,useful for measuring optical properties of tissue in vivo.

FIGS. 7A and 7B are plots of the absorption coefficients.

FIGS. 7C and 7D are plots of the isotropic or reduced scatteringcoefficients of various tissues as a function of wavelength.

FIG. 8A illustrates components of the apparatus used for absolutecalibration determination.

FIG. 8B illustrates how absolute calibration allows determination ofcell count from an animal subject.

FIGS. 9A and 9B illustrate how photons diffuse through tissue (9A) andhow the photons that diffuse to the surface of the tissue are capturedin the invention (9B).

FIG. 10A shows a Monte Carlo simulation following a photon through a“random walk” through turbid tissue.

FIG. 10B illustrates a calculated light intensity spatial profilecalculated for a light source at 4 mm depth from the diffusion model(solid line) and with Monte Carlo simulations (“+” symbols).

FIGS. 11A and 11B are plots calculated from the diffusion model showingpeak intensity (11A) and spot width (FWHM) (10B) as a function of sourcedepth.

FIG. 12A is a surface light-intensity image from a light-emission sourcein a subject, and shows the horizontal and vertical profile lines alongwhich light-intensity profiles were measured.

FIGS. 12B and 12C are light-intensity profiles measured along thehorizontal and vertical profile lines shown in FIG. 12A.

FIG. 13A shows spectral image plots of luciferase light-emitting sourcesacquired in vitro (solid line), from the subcutaneous site shown in FIG.13B (squares); and from the lung sites seen In FIG. 13C (diamonds).

FIGS. 13B and 13C are surface light-intensity images from animals with asubcutaneous light-emitting source (FIG. 13B) and light-emitting sourcesin the lungs (FIG. 13C).

DETAILED DESCRIPTION I. Definitions

Unless otherwise indicated, the terms below are defined as follows:

A “biophotonic function” is a mathematical function that describes ameasurable photonic output, such as a light emission profile, spectralintensity distribution, or integrated light intensity, in terms ofphotonic-source variables, such as source depth, size and shape, numberof light-emitting cells, wavelength-dependent scattering and absorptioncoefficients for specific types of tissue, and spectral characteristicsof the light-emitter. The photonic function can be based, for example,on a photon-diffusion model, as discussed below, on a Monte-Carlosimulation, or finite element analysis.

“Turbid tissue” is non-transparent tissue in a subject that has bothlight scattering and light absorption properties.

“Target region” refers to an internal subsurface region in a subject,such as a subsurface tissue or organ, a solid tumor, or a region ofinfection, that is (i) localized within the subject, (ii) separated fromthe surface region of the subject by turbid tissue, and (iii) preferablypossesses at least one distinguishable feature, such as a tissue ororgan-specific antigen, gene or gene product, either as mRNA orexpressed protein, a product resulting from activation, deactivation, orregulation, of a cell, organ, or tissue.

Light-emitting source refers to a source of light emission at the targetregion. The source may itself emit visible light, such as when thetarget tissue is genetically modified to produce recombinantlight-emitting protein when specific genes are expressed by someactivation means. In preferred embodiments, different types oflight-generating proteins, such as bioluminescent proteins (e.g.,luciferases) and fluorescent proteins (e.g., GFP, dsRed, and YFP) areemployed. Exemplary light-emitting sources include prokaryotic andeukaryotic cells transformed with a light-generating protein andadministered to the animal subject, as well as light-producing cellswhich are intrinsically part of an animal made transgenic for alight-generating reporter gene.

Alternatively, the source may include fluorescent or other moleculesthat can be excited by electromagnetic radiation to emit visible light.For example, a composition effective to cause a target to emit light maybe administered to the subject and allowed to localize at the targetlocation wherein the composition will then emit light, or cause someother moiety in or adjacent the target to emit light. For example, theadministered composition may be a compound which activates cells in oradjacent the target, and in-turn, causes such cells to emit light,hence, the localized cells are the target within the subject's bodyregion. In yet other embodiments, infectious cells are administered to asubject, and the progress and locality of the disease are determined.The target, in this embodiment, is the cluster of cells at a given pointhaving a location within the body region of the subject. In otherembodiments of the invention, a body region may have multiple targets,and a subject may have multiple body regions, each region potentiallyhaving a target.

“Fluorochromes” or “fluorophores” are molecules which are excited at oreffectively near an excitation maxima, and emit or fluoresce light atlonger wavelengths. The difference between the excitation maxima of afluorochrome, and the emission maxima of the fluorochrome, is known as aStokes shift. The greater the Stokes shift for a given fluorochrome, thegreater the difference in between the excitation spectrum, or range ofwavelengths, which excites the fluorochrome, and the emission spectrumor range of wavelengths of light emitted by the fluorochrome when isfluoresces. Certain embodiments of the invention employ fluorochromeswith high Stokes shifts such with Texas Red, rhodamine, Cy3, Cy5, Cy7,and other deep, far red, or near infra-red (NIRF) fluorochromes.Particularly preferred fluorochromes emit light at wavelengths longerthan about 600 nanometers. Other preferred fluorochromes will emit lightin ranges shorter than 600 nanometers. In particular, variants of “greenfluorescent protein” (GFP), each having various emission spectra, areparticularly useful because, like luciferase, such proteins may besynthesized by the target or cells adjacent or associated with thetarget, or which localized at the target, for example, as in aninfection. GFPs may further be administered as a conjugate to anotherprotein or biological material that localizes at the target or targets.

In preferred embodiments of the invention, each light-emitting source,under known light-emitting conditions, has a known emission spectrum.Each emission spectrum is preferably constant throughout the detectionstep. In particular, the relative intensity of light emission at eachwavelength of light remains relative to the intensity of light emissionat all other wavelengths of light within the emission spectrum of thelight-emitting source, as the overall intensity of the spectrum changes.Some embodiments of the invention minimally require at least twodistinguishable ranges of wavelengths of emitted light. More preferably,particular embodiments of the invention require that each range ofwavelengths of light behave differently, as a function of the distanceor depth for which such light travels through the body region, fromother ranges as they travel through a selected body region of a subject.For example, the intensity of a first range of wavelengths of light iscut in half for every centimeter of a body region the light travels,whereas a second range of wavelengths of light is cut by three fourthfor every centimeter of body region the light travels through. Theintensity of light having both ranges of wavelengths, where each rangehas the same intensity, will diminish by one half at the first range,and three quarters at the second range, after passing through onecentimeter of tissue. By comparing the resulting 2:1 ratio ofintensities from the initial 1:1 ratio of intensities at tissue depthzero, a determination that the light traveled one centimeter through thetissue can be made.

The invention may be practiced using different light-emitting sourcessuch as different luciferase enzymes. For example, the light emissionfrom “blue” bacterial luciferase (Photorhabdus luminescens) expressed onagar was analyzed by a spectrometer that revealed a spectrum centeredabout 485 nm. A “green” firefly luciferase (Photinus pyralis) expressedin PC-3M cells in PBS solution emitted light demonstrating a spectrumcentered about 570 nm. A “red” luciferase expressed in PC-3M cellssuspended in PBS solution demonstrated a spectrum centered about 620 nm.

II. Apparatus and Method

FIG. 1A is a cross-sectional view of an apparatus 20 which may be usedin investigating the location and size of a light-emitting source in asubject, in accordance with the invention. The apparatus generallyincludes a light-tight enclosure or chamber 22 within whichlight-emission events from the subject can be detected. Light tight box22 may be constructed as described in co-owned PCT publication number WO200163247, which is herein incorporated by reference in its entirety.Briefly, the chamber is defined by back and side walls, such as backwall 21, and has a front opening (not shown, which can opened toprovided access to the interior of the chamber, and closed to provide alight-tight seal. Although light tight box 22 was designed for smallanimals, the technology can also be applied to larger mammals, includinghumans.

Contained within the chamber is a stage 24 on which the subject isplaced and preferably immobilized during light-emission detection.Although many of the examples described herein have to do with a smallmammalian subject, typically a mouse or a rat, it will be appreciatedthat the same principles and methods will apply to other animals as wellas human subjects, employing suitable scale-up in chamber size and wherenecessary, suitable increases in the size and number of light emittersin the target region. The stage in the apparatus shown is designed to beraised and lowered to a selected vertical position within the chamber bya conventional wormscrew mechanism shown generally at 26 and under usercontrol. The stage position within the chamber may be monitored by anyof a number of known tracking devices that do not require visible lightsignals.

The viewing optics includes to two general components. The first is alens assembly 28 that serves to focus light-emission events from thesurface of the subject onto the detection screen of a photodetector 32,which is typically a photodetector pixel array of a charged-coupleddevice that can be operated in a cooled condition to minimize intrinsicnoise level. The construction of the lens assembly and its opticalcoupling to the detector is conventional. One preferred CCD is a model620 CCD, commercially available from Spectral Instruments.

The second, and optional, component of the optical system is awavelength filter wheel 30 containing a number of optical bandpassfilters designed to block light transmission in all but a selected rangeof visible-light wavelengths. One standard installation includes 3filters; a shortpass filter 30 a, for wavelengths less than 510 nm (bluelight filter), a midpass filter 30 b having a bandpass in the 500-570 nmrange (green light filter), and a longpass filter for wavelengthsgreater than 590 nm (red light filter). In some applications, filterscan provide more precise bandpass, e.g., every 20 nm. The just-describedviewing optics and detector are also referred to herein, collectively,as an optical system.

The apparatus also includes a control unit 34. Various user-controlledsettings, such as stage height, orientation, and translation position,bandpass filter, and detector mode are made through a control inputpane; 36 in the apparatus.

FIG. 1B shows a more schematic representation of apparatus 20, hereshown in a scale for use in imaging a small mammalian subject, such as amouse 38. The lens assembly is represented here as a single lens 28, thefilter wheel at 30, and a CCD detector at 32. The detector output issupplied to a computational unit 40 in the control unit computationalunit 34 which includes a processor 40 and a storage unit 42 for datafiles. The content of the data files, and operation of the computationalunit, in response to light-detection signals from the designs fromdetector 32 will be described below. The control unit is operativelyconnected to a display/output device 44, such as a computer monitor, fordisplaying information and images to the user.

The operation of the computational unit can be appreciated from themethod of the invention carried out by the apparatus, which will besummarized with respect to FIG. 2, and detailed further below. The firststep in the method is to obtain a first measured light-intensityprofile. This is done by first localizing a light-emitting source withina subject. This may be done, as noted above, in a variety of ways,including introducing into the subject, a gene effective to produce alight-emitting protein, such as luciferase, in the target region, orlocalizing a fluorescent compound at the target source, both accordingto methods which are known, e.g., as described in co-owned PCTpublications WO00/36106, WO01/18225, WO00/54581 and WO97/40381; all ofwhich are incorporated herein by reference.

The subject so prepared is placed in the light-tight chamber, andlight-emission form the subject surface originating from light-emissionevents at the target region are measured, digitized, and placed in thecomputational unit, as at 52 in FIG. 2. The computational unit uses thisdata to generate a spatial profile of light emission, as at 54. Thisprofile could be taken along a selected row and a selected column ofdetector elements in the detector representing profiles along x(horizontal) and y (vertical) axes in a plane parallel to the stage inFIG. 1A. That is, the profile is a plot of measured intensity valuesalong a selected row and column of detector elements, and represents thedistribution of light intensity values that are focused from the subjectsurface onto the detector array. Exemplary light-intensity valuesmeasured from a subject are shown in solid lines in FIG. 3B. FIG. 3Ashows the emission contours, and FIG. 3B shows the vertical profile, asdetermined at detector pixels corresponding to those positions.

Turning back now to FIG. 2, the spatial profile or profiles so obtainedare matched or fitted with profiles stored in a database 58 ofparameter-based photonic functions which are either empirical functionsmeasured from light-emitting sources of known size and depth, or arecalculated from photon-diffusion models using depth and size parameters,and optionally, other parameters, as will be described below.

From the optimal curve match, the program can make an initial estimateddetermination of depth and/or size and/or brightness, preferably allthree, of the light-emitting source. According to a feature of theinvention, the method is now carried out in a way that the depth andsize determinations are refined by additional data that may be in theform of additional light-intensity data, and/or additional modelingdata. For example, and as indicated in FIG. 2, the data may be in theform of:

(a) 2nd-view data, as at 62, meaning light-intensity data, typicallyspatial profile data, obtained by viewing the subject from a second viewangle. Typically, the subject in the apparatus is tilted with respect tothe optical system so that light-emission is obtained from a secondregion of the subject surface;

(b) Spectral data, as at 64, meaning light-intensity data obtained atone or more bandpass ranges, e.g., in a blue-light, green-light, orred-light spectral range;

(c) Transmittance data, as at 66, meaning predetermined-light intensityvalues obtained typically as a function of wavelength at selectedlocations in a subject that approximates the subject of interest;

(d) Tissue properties data, as at 68, meaning predetermined values oftissue parameters, such as reduced scattering coefficient μ′_(s),absorption coefficient μ_(a), or effective coefficient μ_(eff)corresponding to the subject tissue through which light is diffusing.This data is used, for example, to refine the spatial profile curvesgenerated by a diffusion model;

(e) Simulation data, as at 70, meaning predetermined light-intensityvalues, typically spatial profile data, obtained by placing alight-source of known intensity and size at selected locations in amodel representing the subject target region and adjacent surface, or inan actual subject that approximates the subject; and

(f) Total intensity data, as at 72, meaning total light intensity summedor integrated over an entire spatial profile or an entire detectorarray.

The program run by the computation device receives the additionalinformation at 60, and uses the information to refine the depth and sizedetermination of the light-emitting source, at 74, as will be consideredbelow. Finally, the refined determination of source depth, size, andoptionally, shape and number of cells in the light-emitting source, isdisplayed to the user, either in the form of data and/or one or moresubject images showing the location and intensity of the light-emittingsource.

(g) Absolute calibrated light intensity, as at 73. Calibration toabsolute intensity allows one to convert counts/sec/pixel in the CCD toradiance photons/s/cm²/sr (sr=steradians). FIG. 8A illustrates anoptical assembly 100 used in the calibration method. The assemblyincludes, in addition to a CCD detector 102, lens 104 (representing alens assembly, as above), a bandpass filter 106 and a low-light-levelintegrating sphere, such as a OL Series 425 sphere available fromOptronic Laboratories, which acts as a source of a known lightintensity. The known radiance from the sphere, measured inphotons/sec/cm²/sr, allows one to calculate the calibration factor thatrelates the two numbers.

FIG. 8B illustrates how absolute measured intensity is used to calculatetotal number of cells in a light-emitting source. It is assumed thateach cell will produce a photon flux φ_(c) photons/sec/cell. Theradiance from a light-emitting source 110 in a subject 112, as measuredby detector 102, measures the number of photons/s/cm²/sr. The measuredradiance is then integrated over a region of interest to convert to asource flux φ_(s) photons/sec. From the known flux values, the number ofcells in the light-emitting source is readily calculated as φ_(s)/φ_(c).

III. Light-Emission Data and Parameters

This section considers various types of light-emission data that may becollected in practicing the invention, and discusses how the data may beused in photon-diffusion modeling, and/or for determining depth or sizeof a light-emitting source based on curve matching with a modeldiffusion curve.

Light-intensity spatial profile. The basic light-emission data that isdetermined is a light-intensity spatial profile which has been describedabove, with reference to FIG. 3B. A measured light-intensity spatialprofile shows the spatial distribution of light intensity emitted fromthe surface region of subject. The light-intensity spatial profile maybe obtained along a single line, along 2 or more lines (e.g., along thex and y directions with respect to the stage supporting the subject).The entire 2D surface distribution of light may also be used. A modeledspatial profile represents the predicted light-intensity spatialdistribution calculated from a model of photon diffusion through aturbid tissue. By matching a measured profile with a modeled profile, anapproximate depth and/or size of light-emitting source can bedetermined.

Total light intensity. Total light intensity is the total lightintensity summed or integrated over all of the detector elements in thedetector. Total light intensity can be used as a further constraint onthe fitting model used to determine source, location, and brightness.

Absolute calibrated light intensity. As described above with referenceto FIG. 8B, the absolute light-intensity flux can be used to measuretotal flux from the light-emitting source. Assuming that this source ismade up of cells which each emit an average cell flux φ_(c), the totalnumber of light-emitting cells making up the source can be determined.This determination, in turn, can be used to refine the total source massor volume determination, based on a known or estimated cell mass ratiofor the tissue source.

Spectral data. Spectral data is light-intensity data collected atparticular wavelength ranges, e.g., the blue-light, green-light, andred-light wavelength ranges noted above. Because light is absorbedpreferentially at shorter wavelengths by subject tissue, andparticularly by hemoglobin in subject tissue, the relative intensitiesof light at different wavelengths, can provide information about sourcedepth and can be used in producing refined wavelength-dependent spatialprofiles, based on wavelength-dependent tissue scattering and absorptioncoefficients.

FIGS. 4A and 4B depict the differential effect distance or depth hasupon light that travels through the tissue of the body region, atdifferent wavelengths or ranges of wavelengths. The graph showsincreasing light intensity on the y-axis and increasing light wavelengthon the x-axis. The solid line in FIG. 4A represents the in vitroemission spectra for the light-emitting source that correlates to azero-depth emission spectrum which comprises a broad single peakcentered about 495 nm. The broad dashed line represents the emissionspectrum of the light-emitting source after it has passed through aboutone millimeter of tissue (in vivo). At about 600 nm, the one millimeterdepth emission spectrum develops a small peak, as the once large peak atabout 495 nm begins to diminish in relative intensity. The intermediatedashed line represents the emission spectrum of the light-emittingsource after it has passed through about two millimeters of tissue (invivo). The 495 nm peak becomes lesser in intensity, while the 600 nmpeak increases and becomes more pronounced. Lastly, the dotted linerepresents the emission spectrum of the light-emitting source after ithas passed through about four millimeters of tissue (in vivo) where the495 nm peak has been further reduced.

FIG. 4B represents the emission spectrum at four millimeters depth oftissue scaled against the zero tissue depth emission spectra. The 600 nmpeak is prominent over the once prominent 495 nm peak. In this example,it is the ratio of the 600 nm peak to the 495 nm peak that providesinformation about the depth of the light-emitting source.

Preferred methods of the invention employ self-scaling computations sothat the depth of the light-emitting source may be determinedindependent of its overall intensity. To achieve this end, functions areformulated that utilize ratios of two or more ranges of wavelengths,where each range of wavelengths travels through tissue in a body regionat different rates as a function of depth of the target from the surfaceof the body region.

Through-body transmittance. Through-body transmittance is measured byplacing a know-intensity light-source at a selected location against orinside a subject, and measuring the light-intensity that is transmittedthrough the subject on multiple surfaces. The transmittance will have astrong spectral component, owing to the strong spectral dependence ofthe scattering and absorption coefficients of different tissues (seebelow). Typically, transmittance values are pre-determined using a modelsubject, by measuring total transmittance at various selected locationsand at each of a number of spectral ranges.

FIG. 5 shows a top view of a subject having a plurality of body regionsindicated by numbers 1-16. In this example, the subject is animmobilized mouse, and each body region corresponds to pre-determinedregions of interest. Prior to depth determinations by the practice ofthe methods of the invention, the subject is optically analyzed todevelop a data file of optical characteristics for each body region. Inthis example, each body region is placed between a spectral light sourceand a spectral detector. Light transmission spectra are obtained foreach body region. The result is analogous to placing each body region ina spectrophotometer cuvette and measuring the spectral profile of eachregion at different wavelengths and wavelength ranges, to create a datafile of how light transmits through different body regions, and thetissues within such regions. The device of FIG. 1 may be used to developsimilar data for particular ranges of wavelengths provided the device isadapted to trans-illuminate, with respect to the detector, the subjectat each body region tested. Thus, the device of FIG. 1 becomes, inessence, a whole animal spectrophotometer.

FIGS. 6A and 6B show the empirical spectral data gathered from thespectral analysis described immediately above. As is described below inmore detail, this data, either as whole spectra, or particular rangeswithin each spectrum, can be used to create the data files forcomparison with detected intensity information, or for developingfunctions to compute target depth.

In a more detailed approach, the apparatus provides a whole body scannerfor scanning an entire subject, or portions thereof, which furthercomprises a scanning two-color laser for scanning the body of a subject.The scanner moves through an arc laser light of at least two colors,either simultaneously, or sequentially. As the laser light beams upward,it penetrates the body of a subject placed within a rotational tube. Thedetector placed opposite the laser light source is adapted to receiveand measure the laser light as it moves through its arc path.

Tissue optical properties. There are two important optical properties ofsubject tissue used in photon diffusion modeling. The first is theabsorption coefficient μ_(a) of the tissue, which is related to thefraction of incident light that is absorbed per unit pathlength. As seenin FIGS. 7A and 7B, which plot absorption coefficients for a variety oftissue types over the visible spectrum, the absorption coefficient ishighly dependent on the nature of the tissue, and the wavelength oflight, with each tissue showing a peak absorption in the 500-600 nmrange. The relatively low absorption coefficient above about 600 nm isconsistent with the spectral data seen in FIG. 13A, and with the totaltransmittance data shown in FIGS. 6A and 6B.

The second important optical property is the reduced scatteringcoefficient μ′_(s) of the tissue, which is the fractional decrease inintensity per unit length of penetration of light due to large anglelight scattering. As seen in FIGS. 7C and 7D, which plot reducedscattering coefficient for the same tissues over the visible spectrum,scattering effects can be relatively different in different tissues. Ingeneral, the reduced scattering coefficient decreases somewhat at longerwavelengths.

A double integrating sphere (DIS) system is a widely used tool tomeasure the optical properties of tissue or any turbid medium since itconveniently measures the diffuse reflectance, R_(d), and the diffusetransmittance, T_(d), simultaneously. FIG. 6C illustrates the doubleintegrating sphere apparatus 74 used to measure these values in order toobtain optical properties such as the absorption coefficient, μ_(a), andthe reduced scattering coefficient, μ′_(s) (Prahl, S. A., et al.,Applied Optics 32:559-568 (1993); Pickering, J. W., et al., AppliedOptics 32:399-410 (1993)). The sample 76 is placed between the twointegrating spheres 78 and 80, with internal baffles 82 positioned toprevent measurement of directly reflected or transmitted light. Thespheres are coated with a highly reflective material to minimizeabsorption of light. Light is detected with detectors 84 and preferablyanalyzed using a computer 86. Sample illumination between 400-1000 nm isachieved using an arc lamp connected to a monochromator 88, via a fiberoptic cable 90 and lens 92.

From diffuse reflectance, R_(d), and diffuse transmittance, T_(d),values, the absorption and the reduced scattering coefficients areobtained using the inverse adding-doubling program(http://omlc.ogi.edu/software/iad/index.html). This program is anumerical solution to the one speed radiative transport equation, whichdescribes light propagation at steady state in a scattering medium(Prahl, S. A., et al., Applied Optics 32:559-568 (1993)). The program isan iterative process, which estimates the reflectance and transmittancefrom a set of optical parameters until the calculated reflectance andtransmittance match the measured values.

A characteristic of the DIS system is the fact that tissues need to beextracted before the measurement. Therefore, it is desirable to maintaintissue viability under measurement conditions. Vascular drainage ofhemoglobin and tissue hydration need to be considered, especially atwavelengths where hemoglobin and water absorption are high. Partly inview of the foregoing, a preferred method of measuring tissue opticalproperties is a non-invasive in vivo measurement, as follows.

A radial fiber probe similar to that described by Bevilacqua(Bevialcqua, F., et al., Applied Optics 38:4939-4950 (1999)) may be usedto measure optical properties non-invasively or with minimalinvasiveness in vivo. FIG. 6D illustrates an experimental set up of aradial fiber probe 94. Fiber probe 94 includes an illumination source95, at least one illumination fiber 96 and several (e.g., six) detectionfibers 97 generally within a 1-2 mm distance. The probe is then placeperpendicularly on a tissue of interest 99, preferably with minimalcontact pressure. The output of the detection fibers 97 is fed into aset of corresponding detectors 98. The optical properties are spatiallyresolved from intensity versus radial distance from the source. At smallsource-to-detector separations, simple analytical models such as thediffusion approximations are not well suited to describe the systemtherefore Monte Carlo methods are typically used to analyze the data.

IV. Photon Transport Models

This section considers photon-transport models for simulating photonemission at the surface of a body from a light source below the surfaceand moving through a turbid medium. In particular, it is desired togenerate simulated light-intensity spatial profiles based on (i) depthof the light-emitting source, (ii) size of the light-emitting source,and (iii) light-absorption and light-scattering coefficients. Since thelight-absorption and light-scattering coefficients are dependent both onwavelength and on the nature of the tissue through which the photonsdiffuse, the model may be refined to specifically consider the nature ofthe tissue and the spatial profiles at selected wavelengths.

The photon-diffusion model employed herein makes a number of simplifyingassumptions for purposes of generating an initial light-intensityspatial profile from which initial source-depth and source-sizeinformation can be generated, as will be considered in Section V. Themodel may then be expanded by considering additional tissue-dependentand/or wavelength dependent information to refine the spatial profile,for purposes of improving the curve fit with a measured spatial profile.Alternatively, the initial depth and size information can be refined byother types of curve or data matching. Both approaches for refining theinitial depth/size approximation will be considered in Section V below.

FIG. 9A shows a photon path as it diffuses through a turbid tissueformed of cells, such as cell 111 containing organelles 113, As seen,the cell size, typically in the 20-30 micron size range, is largerelative to the wavelength λ of light, which is between about 0.4 and0.6 microns. Scattering is due to abrupt discontinuities (<<λ) atmembranes and is characterized by an inverse length scatteringcoefficient μ_(s) of about 10-20 mm⁻¹. The scattering anisotropy g isabout 0.9, which gives a reduced scattering coefficient μ′_(s)=μ_(s)(1-g), or about 2 mm⁻¹. The absorption coefficient μ_(a) (A) is between0.01 and 1 mm⁻¹, and as seen above, has a strong wavelength dependence.The absorption and scattering coefficients are wavelength-dependent, butfor λ>˜600 nm, it is generally true that μ_(a)<<μ′_(s) in tissue.

A quantitative description of transport of light from a target sitewithin a body to a light detector located adjacent a body surface may beachieved by a solution of a radiative transfer diffusion equation oflight with appropriate boundary conditions (e.g., R. C. Haskel; et al in“Boundary conditions for the diffusion equation in radiative transfer”,J. Optical Soc Am., 11A:2727 (1994)). One approach, for example, uses anextrapolated boundary condition shown schematically in FIG. 9B in whichoptical fluence vanishes at a planar surface that is displaced from thephysical boundary at a distance z_(b) that takes account of the Fresnelreflection of light at the physical boundary.

As a first approximation, the body in which the light-emitting source islocated is represented as a semi-infinite, homogenous turbid medium thatis both scattering and absorbing. The approximation is illustrated inFIG. 9B which shows a light-emitting point source 114 in slab 116 adistance d below the slab surface. The diffusion equation is

D∇ ²φ(r)=μ_(a)φ(r)−S(r)  (1)

and Fick's law is

j(r)=−D∇φ(r)  (2)

where Φ is the isotropic fluence (watts/m²), j is the small directionflux (watts/m²), S is the power density (watts/m³), r is the radius, and

$\begin{matrix}{D = \frac{1}{3\lbrack {{( {1 - g} )\mu_{s}} + \mu_{a}} \rbrack}} & (3)\end{matrix}$

The Green's function solution for a point source P (watts) is

$\begin{matrix}{{{\varphi (r)} = {\frac{P}{4\pi \; {Dr}}{\exp ( {{- \mu_{eff}}r} )}}}{where}{\mu_{eff} = \sqrt{3{\mu_{a}( {\mu_{s}^{\prime} + \mu_{a}} )}}}{and}{\mu_{s}^{\prime} = {( {1 - g} ){\mu_{s}.}}}} & (4)\end{matrix}$

The solution to the diffusion equation in slab geometry using theextrapolated boundary condition is obtained by summing contributionsfrom the source 114 plus image source 118 (as shown in FIG. 9B),resulting in the following equation for radiance at the surface (viewingperpendicular to the surface) for a point source P (watts) at a depth d.

$\begin{matrix}{{L_{z = 0} = {\frac{1}{4\pi}( \frac{P}{4\pi \; D} )\begin{Bmatrix}{\frac{\exp ( {{- \mu_{eff}}r_{1}} )}{r_{1}} - \frac{\exp ( {{- \mu_{eff}}r_{2}} )}{r_{2}} +} \\{3{D\begin{bmatrix}{{\frac{d}{r_{1}^{2}}( {\mu_{eff} + \frac{1}{r_{1}}} ){\exp ( {{- \mu_{eff}}r_{1}} )}} + {\frac{d + {2z_{b}}}{r_{2}^{2}} \times}} \\{( {\mu_{eff} + \frac{1}{r_{2}}} ){\exp ( {{- \mu_{eff}}r_{2}} )}}\end{bmatrix}}}\end{Bmatrix}}}\mspace{79mu} {where}\mspace{79mu} {{r_{1} = \sqrt{\rho^{2} + d^{2}}},\mspace{79mu} {r_{2} = \sqrt{\rho^{2} + ( {d + {2z_{b}}} )^{2}}}}} & (5)\end{matrix}$

ρ is the radius on the slab surface and

$\begin{matrix}{z_{b} = {\frac{1 + R_{eff}}{1 - R_{eff}}\frac{2}{3( {\mu_{a} + \mu_{s}^{\prime}} )}}} & (6)\end{matrix}$

and R_(eff) is the effective reflection coefficient which is about 0.43for tissue.

Plotting surface radiance as a function of p gives the plot shown in thesolid line in FIG. 10B, which has the same derivation as thelight-intensity profile plot shown in dotted lines in FIG. 3B.

The spatial profile curve for a light-emitting point in a turbid mediumwas also calculated using a much more computationally intensive MonteCarlo simulation, which follows each photon through a random walk, asillustrated in FIG. 10A. The Monte Carlo simulation, shown in “+”symbols in FIG. 10B closely matches the spatial profile calculated fromthe diffusion equation.

To illustrate how the diffusion equation may be used to model photondiffusion through various tissues and a various depths, the spatialprofile was calculated for various values of μ_(eff), corresponding toμ_(a) and μ′_(s) values of 2.0 and 20 cm⁻¹, respectively, for μ_(eff)=11cm⁻¹; 0.4 and 10 cm⁻¹, respectively, for μ_(eff)=3.5 cm⁻¹, and 0.05 and5 cm⁻¹, respectively, for μ_(eff)=0.87 cm⁻¹. The three values of forμ_(a) correspond roughly to tissue absorption for blue, green, and redwavelengths.

The plots in FIGS. 11A and 11B show that intensity decreases and spotwidth increases with increasing depth, and that large values of μ_(eff)result in large attenuation of peak intensity with narrower spot width.

V. Determining Depth and Size Information

In practicing the method of the invention, the subject is initiallytreated to localize light-emitting molecules at a target source, asdetailed above. The subject is then placed at a selected location withinthe light-tight chamber of apparatus 20 (appropriately scaled forsubject size) and the optical system is adjusted to measure lightemission events from the source at the surface region of the subjectbetween the light-emitting source and the detection optics. With thesubject immobilized at a selected optical perspective, a light-intensityspatial profile of surface light emission is obtained, also as describedabove. FIG. 3A is a surface map of measured light intensity from asubsurface light source, which was subsequently used to generate thespatial profile shown in FIG. 3B.

The spatial profile(s) are then fitted with parameter-based biophotonicfunctions that relate light-emission intensity to depth of alight-emitting source (and in some cases, source size) to obtain aninitial determination of source depth (and in some case, source size).One preferred biophotonic function is that derived from the simplifieddiffusion model discussed in Section IV above, in which light intensityfrom a point source or source having a defined spherical or ellipsoidalvolume is calculated as a function of source depth, fixed scattering andabsorption coefficients, and surface distance r from the source.Conventional non-linear least-squares curve fitting techniques, such asLevenberg-Marquardt (“Numerical Recipes in C”, Press et al., eds,Cambridge Press, NY, 1989) are suitable for the curve fitting. Curvefitting can be done using a single 1-dimensional (“1-D”) profile, asshown in FIG. 3, a plurality of such profiles, or the entire 2-D spatialdistribution (e.g., as shown in FIG. 12A). Curve-fitting calculations tothe data shown in FIG. 3B (dashed line) indicate a depth of 2.7 mm,which compares well with the estimated actual depth of 2.2 mm.

FIGS. 12A-12C give another example of how depth and size information canbe determined from spatial profiles. The light-emitting source here is asubcutaneous elliptical tumor having actual dimensions in the horizontaland vertical directions of 2.7 mm and 3.2 mm, respectively, and a tumorthickness of 1.5 mm. The horizontal and vertical profiles were (FIGS.12B and 12C, respectively) were fitted with curves generated for anellipsoid light-emitting source having horizontal and verticaldimensions of 1.3 and 2.4 respectively, a thickness of 1.5 mm, and adepth of 0.4 mm.

The source depth (and optionally, source size) determination from theinitial curve fitting can be refined, and source size approximated, byemploying additional data whose effect is to refine the parameters ofthe biophotonic function. The nature of the additional data, and themanner in which it refines the parameters, e.g., depth and size, of thebiophotonic function, providing refined depth and source-sizeinformation, will now be considered for each of the information typesoutlined in Section II above, with respect to FIG. 2.

A. 2nd-View Information.

To obtain 2nd-view data, the subject is rotated with respect to theviewing and detection optics for viewing the light-emitting source fromanother perspective. From this second perspective, a second lightintensity profile is obtained, providing a second depth determinationwith respect to a second subject surface region. By determining theintersection of the source depths at two different surface locations, amore accurate depth and/or source size can be determined. The more viewsthat are obtained, the more accurately depth and source size can bedetermined.

B. Spectral Data.

A target's depth within a body region may be determined, or the targetdepth refined, by (i) causing the target to emit light at two or morewavelength ranges, and (ii) comparing the differences in each range'slight transmission through the body region, at a surface of the bodyregion, provided that the light in each wavelength range transmitsthrough the body region differently from the light in other wavelengthranges as a function of depth.

Typical spectral curves (representing total light intensity at theindicated wavelengths) as a function of source depth are shown in FIG.4A, discussed above. As seen particularly in FIG. 4B (where the peaksare scaled to the in vitro spectral curve), the ratio of peak height atabove 600 nm, e.g., 620 nm, to that the in vitro (zero-depth) peakheight, e.g., around 500 nm, increases dramatically as a function ofdepth. Thus, by generating a spectral curve and determining the ratio ofpeak heights, typically above and below 600 nm, e.g., 620 nm: 500 nm,accurate depth information based on the wavelength-dependent loss inintensity in turbid tissue, can be determined.

A useful formula for determining approximate depth from spectralmeasurements can be derived from Eq. 4 as follows:

$\begin{matrix}{d \approx \frac{\ln ( {\varphi_{1}{D_{1}/\varphi_{2}}D_{2}} )}{\mu_{{eff}\; 2} - \mu_{{eff}\; 1}}} & (7)\end{matrix}$

where d represents depth, φ represents the measured light intensity,μ_(eff) represents the empirically determined effective coefficient ofattenuation, and D is the empirically determined diffusion coefficient.The subscripts 1 and 2 in the above formula refer to two separatewavelengths at which measurements are made.

Using the above formula, depth may be determined from two or more lightintensity measurements at different wavelengths or ranges ofwavelengths. In one embodiment, the depth may be obtained by executingthe following series of steps: (1) Image the bioluminescent cells invitro as well as in vivo (in the animal) at two wavelengths; (2)Quantify the images by measuring, e.g., the peak intensity or theaverage (integrated) intensity for each image; (3) Calculate the ratioof the in vivo image data to the in vitro image data at each wavelength;and (4) Calculate the depth using Equation 7 and an effective scatteringcoefficient μ_(eff) obtained, e.g., from tissue property measurements.An application of this approach is illustrated in Example 1.

In one embodiment, it is preferable to have a significantly differentvalue for each μ_(eff). Animal tissue provides such a difference inμ_(eff), due largely to the presence of hemoglobin, which has a largeabsorption peak just below 600 nm, and relatively low absorption above600 nm.

Alternatively, the additional spectral data may include one or morespatial profiles taken at one or more selected wavelengths or wavelengthranges. The profile(s) are then compared with model intensity functionsgenerated, for example, from a photon diffusion model above, usingwavelength-specific values for the absorption, scattering, and/oreffective coefficients, as described above with respect to FIG. 3B.

In still another application, intensity values can be measured atdiscrete wavelengths, spaced every 20 nm. The spectral plots in FIG. 13Ashow green luciferase in cells in PBS solution, and spectral curves forgreen luciferase localized subcutaneously at a site indicated in FIG.13B and in the lungs as shown in FIG. 13C. All the curves are normalizedto a value of 1 at 700 nm. The ratio of intensities at 560 nm to 620 nmis indicative of depth.

As noted above, a related aspect of the invention provides a method ofdetermining the depth of a light-emitting source in a subject. Inpracticing the method, the light emission intensity from the subject attwo or more different wavelengths between about 400 and about 1000 nm.The depth of the light-emitting source is determined using the measuredlight-emission intensities, and information related to the opticalproperties of the subject, e.g., coefficients of attenuation anddiffusion in the subject.

Information relating to the optical properties, .e.g., coefficients ofattenuation and diffusion in the subject may be obtained by directmeasurement of the coefficients in material that is the same or similarto that of the subject. This information, combined with measuredintensities at two wavelengths in vitro (zero depth) and in vivo (thedepth to be determined) can be applied to Equation 7 to determine thedepth of the light source, as described above, and as illustrated inExample 1.

Alternatively, optical-properties information may be obtainedindirectly, by determining, at two or more different wavelengths, lightintensities from a light-emitting source located at each of a two ormore of a plurality of depths in tissue or material corresponding tothat of the subject. The desired depth determination is then made bymatching the measured light intensities, e.g., the ratio of theintensities at the different wavelengths, with the light intensitiesdetermined at each of the plurality of depths. In a more specificapproach, the spectral profile of light intensities from thelight-emitting source may be compared (matched) with each of a pluralityof spectral profiles of light intensities from a light-emitting sourceat each of a plurality of depths.

C. Through-Body Transmittance Data.

Through-body transmittance data is obtained as described above, andillustrated in FIGS. 5, 6A and 6B for a small-animal subject. As notedabove, the information provides a wavelength dependentlight-transmission value through a selected tissue or tissues of a knownthickness. Predetermined transmittance data from a number of selectedlocations in a subject can be used to estimate average whole-bodyscattering and absorption coefficients at the different subjectlocations, for purposes of refining the modeled spatial profile, eitherover the entire visible spectrum, or at selected wavelengths, used forcurve fitting to the measured spatial profile.

D. Tissue Properties Data.

The tissues property data, such as that shown in FIGS. 7A-7D, typicallyincludes wavelength dependent absorption and scattering coefficients foreach of the major body tissues. This data is used, for example, in theabove model of photon diffusion, to refine the spatial profiles thatwould be produced by light transmission through a given tissue, at agiven wavelength, typically in the red wavelength. Thus, for example, ifthe light-emitting source in the subject is muscle, and the spatialprofile is taken in the red wavelength, a refined spatial profilegenerated with the tissue-specific and wavelength-specific absorptioncoefficient will provide a refined spatial profile curve for curvefitting with the measured curve.

E. Simulation Data.

In another embodiment, a test point or otherwise known-shapelight-emitting source may be introduced into a block or slab thatsimulates turbid tissue. The block can be prepared with variousscattering and diffusion coefficients, and various shapes to simulatelight-emitting source conditions in a subject. Upon placement of thetest point, spectral profiles may be taken from the outer surface of thebody region where the test point was placed. This data then iscorrelated with the actual depth and location of the test point. Bymoving the test point from point to point within the block, a series ofspectral measurements can be made. From this series of spectralmeasurements, a data file can be assembled to model the spectralresponses of different regions within the subject.

F. Integrated Light Intensity

Summed or integrated light-intensity, as noted above, refers to thelight-intensity summed over all or some defined area of the detectorarray. The integrated light intensity can be compared with the integralof Equation 5 to provide yet another estimate of source depth andbrightness. This information can be used in conjunction with the profileinformation. The integrated light intensity can also be calculated formultiple wavelengths.

G. Calibrated-Intensity Data.

In the absence of calibrated intensity data, intensity measurements mayvary from camera to camera, and measured values will depend on variablesuch as field of view, binning, time, and f-stop.

Absolute calibrated intensity (Section III above), allows one to refinethe depth-of-source determination, based on curve fitting to true peakvalue, and to estimate the number of bioluminescent cells in tissue, asdiscussed in Section III above.

In still other embodiments, the invention provides for the integrationof other imaging data with the intensity and spatial distribution datato produce greater detail three-dimensional maps of the subject andtarget located therein. For example, compiled differential lighttransmission data, such as disclosed above, may be interlaced withcoordinate systems derived from other three dimensional imaging systemssuch as stacked slices of MRI images forming a three dimensionaldigitized “image” or coordinate system. Such a coordinate system maythen be used to better illustrate the anatomy of the subject for which atarget is being identified. In the case where the subject is alaboratory animal such as a rat, such animals are highly uniform instructure from one animal to the next for a given strain. Consequently,a coordinate data, spectral data, and spatial intensity pattern filesmay be developed by a commercial vendor, and sold for use withsimplified detection units such as shown in FIG. 1. If modelthree-dimensional information is supplied as a data file, then userunits do not need to be equipped for three-dimensional scanning andmapping. The two-dimensional images contemplated in the methods andapparatuses disclosed above may be combined with the three-dimensionaldata files provided by the vendor to yield complete three dimensionalinformation about the size, shape, depth, topology, and location of atarget within the subject.

The following examples illustrate, but in no way are intended to limitthe present invention.

Example 1 Calculating Depth of a Light-Emitting Object UsingTwo-Wavelength Spectral Images

The spectral information shown in FIG. 13A was used to calculate thedepth of luciferase-labeled cells in an animal that received asub-cutaneous injection of the cells (FIG. 13B) and an animal havinglabeled cells in its lungs (FIG. 13C). The analysis was performed usingdata at wavelengths equal to 600 nm and 640 nm using the followingsteps: (i) the bioluminescent cells were imaged in vitro as well as invivo at 600 nm and 640 nm; (ii) the images were quantified by measuringthe average intensity for each image; (iii) the ratio of the in vivoimage data to the in vitro image data were determined at eachwavelength; and (iv) the depth was calculated using Equation 7 and anaverage effective scattering coefficient μ_(off) estimated from theabsorption and scattering coefficients in FIG. 7.

For the subcutaneous cells, the ratios of in vivo to in vitrointensities (relative intensities, or 0) are 0.35 and 0.75 at 600 nm and640 nm, respectively. For the lung signals, these same ratios are 0.05and 0.47. Using μ_(a)=0.25 mm⁻¹ and μ_(s)′=1.0 meat 600 nm andμ_(a)=0.05 mm⁻¹ and μ_(s)′=1.0 meat 640 nm results in μ_(eff)=0.97 mm⁻¹at 600 nm and 0.4 mm⁻¹ at 640 nm. The values of the diffuse coefficientD are 0.27 mm and 0.32 mm at 600 nm and 640 nm respectively.Substituting these numbers into Equation 6, reproduced below,

$d \approx \frac{\ln ( {\varphi_{1}{D_{1}/\varphi_{2}}D_{2}} )}{\mu_{{eff}\; 2} - \mu_{{eff}\; 1}}$

(in this case, subscript 1 refers to 600 nm and subscript 2 refers to640 nm), results in d=1.6 mm and d=4.0 mm for the subcutaneous and lungdepths, respectively.

Although the invention has been described with respect to particularembodiments and applications, it will be appreciated that variouschanges and modification can be made without departing from theinvention.

1. An imaging system, comprising: a light-tight enclosure configured toenclose a separate imaging subject having an internal light emittingsource contained therein; an optical system situated proximate thelight-tight enclosure and configured to measure the light emissionintensities of the light emitting source within the imaging subject attwo or more significantly different wavelengths; and a computationalunit operatively connected to the optical system and configured togenerate a model with respect to one or more properties of the lightemitting source based upon the measured light emission intensities,wherein said computational unit is further configured to refine themodel using data in addition to the measured light intensities.
 2. Theimaging system of claim 1, wherein the one or more properties of thelight emitting source are selected from the group consisting of anapproximate depth, shape, and brightness of the source.
 3. The imagingsystem of claim 1, wherein the optical system includes acharged-coupled-device that can be operated in a cooled condition, and alens for focusing light onto the charged-coupled-device.
 4. The imagingsystem of claim 1, wherein the optical system is designed to detectphotons emitted from a plurality of different selected surface regionsof interest of the imaging subject.
 5. The imaging system of claim 1,wherein the optical system includes one or more wavelength filtersadapted to transmit photons within different selected wavelength ranges,for use in measuring photon-emission intensities within two or moredifferent selected wavelength ranges between about 400-1000 nm.
 6. Theimaging system of claim 5, wherein the computational unit is furtherconfigured to determine the relative light intensities measured at thedifferent wavelengths and compare the determined relative lightintensities with known relative signal intensities at said differentwavelengths as a function of tissue depth.
 7. The imaging system ofclaim 5, wherein the computational unit is further configured to comparethe measured spectrum with a plurality of spectra measured from lightemitting sources placed at various depths within tissue and determinethe depth of the light emitting source from matching the measuredspectrum with the known spectra.
 8. The imaging system of claim 1,wherein the computational unit is further configured to generate avisual 2-, or 3-dimensional representation of the light emitting sourceusing the refined model, and to superimpose the visual representationonto a 2- or 3-dimensional image of the imaging subject.
 9. A method ofinvestigating the location and light intensity of a light emittingsource within an imaging subject, comprising: measuring light emissionintensity from the imaging subject at two or more significantlydifferent wavelengths; obtaining information related to opticalproperties of the material of the imaging subject; using the measuredlight intensities and the obtained information to model one or moreproperties of the light emitting source; and refining said model of theone or more light emitting source properties using data in addition tothe measured light emission intensity and the obtained informationrelated to optical properties of the material of the imaging subject.10. The method of claim 9, wherein the one or more properties of thelight emitting source are selected from the group consisting of anapproximate depth, shape, and brightness of the light emitting source.11. The method of claim 9, wherein said step of measuring involves usingone or more wavelength filters adapted to transmit photons withindifferent selected wavelength ranges.
 12. The method of claim 11,wherein the different selected wavelength ranges are between about400-1000 nm.
 13. The method of claim 9, further including the steps of:determining the relative light intensities measured at the differentwavelengths; and comparing the determined relative light intensitieswith known relative signal intensities at said different wavelengths asa function of tissue depth.
 14. The method of claim 9, further includingthe steps of: comparing the measured spectrum with a plurality of knownspectra measured from light emitting sources placed at various depthswithin tissue; and determining the depth of the light emitting sourcefrom matching the measured spectrum with the known spectra.
 15. Themethod of claim 9, further including the steps of: generating a visual2-, or 3-dimensional representation of the light emitting source usingthe refined model; and superimposing the visual representation onto a 2-or 3-dimensional image of the imaging subject.
 16. A non-transitoryprogram storage device readable by a machine tangibly embodying aprogram of instructions executable by the machine to perform a method ofinvestigating the location and size of a light emitting source in animaging subject, comprising: computer code for measuring light emissionintensities from the imaging subject at two or more significantlydifferent wavelengths; computer code for obtaining information relatedto optical properties of the material of the imaging subject; computercode for using the measured light intensities and the obtainedinformation to model one or more properties of the light emittingsource; and computer code for refining said model of the one or morelight emitting source properties using data in addition to said firstmeasured light intensities and said obtained information related tooptical properties of the material of the subject.
 17. Thenon-transitory program storage device of claim 16, wherein the one ormore properties of the light emitting source are selected from the groupconsisting of an approximate depth, shape, and brightness of the lightemitting source.
 18. The non-transitory program storage device of claim16, further including: computer code for determining the relative lightintensities measured at the different wavelengths; and computer code forcomparing the determined relative light intensities with known relativesignal intensities at said different wavelengths as a function of tissuedepth.
 19. The non-transitory program storage device of claim 16,further including: computer code for comparing the measured spectrumwith a plurality of known spectra measured from light emitting sourcesplaced at various depths within tissue; and computer code fordetermining the depth of the light emitting source from matching themeasured spectrum with the known spectra.
 20. The non-transitory programstorage device of claim 16, further including: computer code forgenerating a visual 2-, or 3-dimensional representation of the lightemitting source using the refined model; and computer code forsuperimposing the visual representation onto a 2- or 3-dimensional imageof the imaging subject.